It is well established that embryonic stem (ES) cells can differentiate into functional cardiomyocytes in vitro. ES-derived cardiomyocytes could be used for pharmaceutical and therapeutic applications, provided that they can be generated in sufficient quantity and with sufficient purity. To enable large-scale culture of ES-derived cells, we have developed a robust and scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, stirred 2 L bioreactor following inoculation with a single cell suspension of mouse ES cells. Utilizing a pitched-blade-turbine, parameters for optimal cell expansion as well as efficient ES cell differentiation were established. Optimization of stirring conditions resulted in the generation of high-density suspension cultures containing 12.5 x 10(6) cells/mL after 9 days of differentiation. Approximately 30%-40% of the EBs formed in this process vigorously contracted, indicating robust cardiomyogenic induction. An ES cell clone carrying a recombinant DNA molecule comprised of the cardiomyocyte-restricted alpha myosin heavy chain (alphaMHC) promoter and a neomycin resistance gene was used to establish the utility of this bioprocess to efficiently generate ES-derived cardiomyocytes. The genetically engineered ES cells were cultured directly in the stirred bioreactor for 9 days, followed by antibiotic treatment for another 9 days. The protocol resulted in the generation of essentially pure cardiomyocyte cultures, with a total yield of 1.28 x 10(9) cells in a single 2 L bioreactor run. This study thus provides an important step towards the large-scale generation of ES-derived cells for therapeutic and industrial applications.

Confocal laser scanning microscopy (CLSM) is being increasingly used for observing protein uptake in porous chromatography resins. Recent CLSM studies have revealed the possible existence of a nondiffusive protein transport mechanism. Observing protein uptake with CLSM requires labeling the protein with a fluorescent probe. This study examines the effect of the probe identity on the subsequent CLSM adsorption profiles. The adsorption of lysozyme conjugated with different fluorescent probes (Cy5, BODIPY FL, Atto 635, and Atto 520) on SP Sepharose Fast Flow was measured using CLSM and zonal chromatography experiments. Results from zonal chromatography show that the retention time of lysozyme-dye conjugates differ significantly from unlabeled lysozyme. The change in retention of lysozyme upon conjugation with a fluorescent probe is consistent with the difference in net charge between the lysozyme-dye conjugate and unlabeled lysozyme. The adsorption profiles measured by CLSM show significantly different behavior depending upon whether the lysozyme-dye conjugate is retained longer or shorter than the unlabeled lysozyme. These results strongly suggest that the lysozyme concentration overshoot observed in previous CLSM experiments is the result of displacement of weaker binding labeled lysozyme by stronger binding unlabeled lysozyme.

Exploiting embryonic stem cell (ESC)-derived cardiomyocytes as a vital source for cell therapies and tissue engineering will depend on robust, large-scale production processes. Recently, we have reported stirring-controlled formation of embryoid bodies, enabling the generation of pure cardiomyocytes in 2-L scale. Expansion and differentiation of genetically engineered mouse ESCs was followed by antibiotic-based cardiomyocyte enrichment. Here we have investigated modification of various parameters aiming at process optimization in a 250-mL spinner flask system. Duration of the differentiation phase, timing of retinoic acid addition, and a slower medium exchange rate were found to be crucial to enhancing cardiomyocyte yield. Improved process conditions were consequently transferred to a 2-L controlled bioreactor. Employing a manual fill-and-draw medium change resulted in the formation of 0.86 x 10(9) cardiomyocytes in a single 2-L batch, thereby reproducing our previous findings. In contrast, an automated perfusion-based strategy enabled the production of 4.6 x 10(9) cardiomyocytes in a single run. This is significantly higher than previously reported and highlights the enormous process optimization potential in the scalable generation of ESC-derived cell lineages.

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